Faculty Bibliography

The 2'-5' oligoadenylate synthetase (OAS) represents a family of interferon-induced proteins which when activated by double-stranded (ds) RNA polymerizes ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)n, n> or =1. Three forms of human OASs have been described corresponding to proteins of 40/46, 69/71 and 100 kDa (p40/p46, p69/p71 and p100). Polyclonal antibodies raised against p100 cross reacted with the other forms p40/p46, p69/p71 but also with an interferon-induced 56-kDa protein (p56). By screening a cDNA expression library, these polyclonal antibodies selected a cDNA encoding p56. Further studies by the RACE procedure using primers corresponding to this cDNA, a p56-related protein of 30 kDa (p30) was isolated. Both p56 and p30 mRNA are expressed in interferon-treated cells as transcripts of 2 kb and 1.8 kb, respectively. The 1.8-kb mRNA is homologous to the 2-kb mRNA but with a 243-nucleotide deletion at position 1011, which results in a frameshift. Consequently, the p56 and p30 have their first 219 amino acid residues identical but differ at their C-termini. In vitro transcription-translation of p56 and p30 cDNAs generated proteins of 56 and 30 kDa, respectively. The deduced amino acid sequence of p56 kDa shares strong similarity with the previously cloned OASs, and contains the subdomains conserved in p40/p46 and p69/p71 forms. Transient expression in HeLa cells indicated that p30 has a cytoplasmic localization, whereas p56 has cytoplasmic and nucleolar localizations. The p56 isolated from transfected cells was shown to bind dsRNA and DNA, but it was devoid of 2'-5'OAS activity typical of the three known forms of this enzyme. Thus, p56 and p30 are two related and interferon-induced proteins outside the family of 2'-5'OAS, which might have as yet unidentified catalytic activities or functions.

The 2',5'-oligoadenylate synthetases (OAS) represent a family of interferon-induced proteins implicated in the mechanism of the antiviral action of interferon. When activated by double-stranded RNA, these proteins polymerize ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)n, n >/= 1. Three forms of human OAS corresponding to proteins of 40/46, 69/71, and 100 kDa have been described. Based on the deduced amino acid sequences of the corresponding cDNAs, these OAS share a homologous region of about 350 amino acid residues that could represent the functional domain of OAS; the 40/46 proteins contain one single domain, whereas the 69/71- and the 100-kDa proteins contain two and three adjacent domains, respectively. Here we show that the cDNAs for OAS-40/46, OAS-69/71, and OAS-100 hybridize to distinct interferon-induced mRNAs of 2 kb; 2.8, 3.3, 3.9, and 4.5 kb; and 7 kb, respectively. By in situ hybridization, we assign the human OAS-40/46, OAS-69/71, and OAS-100 genes (referred to as OAS1, OAS2, and OAS3, respectively) to a unique cytogenetic location on chromosomal region 12q24.2. We constructed a YAC, PAC, and cosmid contig carrying the three OAS genes and provide evidence that the three genes are clustered within a single PAC clone of 130 kb. The three OAS genes are flanked by markers WI-10614 (cen) and D12S2293 (tel) and are contained within three sets of overlapping cosmid clones. They share the same orientation of transcription and are arranged in the order cen- 5'-OAS1-OAS3-OAS2-3'-tel. We suggest that clustering of these genes reflects their evolutionary relationship possibly through the duplication of the conserved functional domain. This ready-to-sequence PAC and cosmid contig provides a valuable tool for identifying regulatory elements involved in the transcription of the OAS genes when induced by interferon and for elucidating the exon-intron organization of these genes.

The protein kinase PKR and the 2',5'-oligoadenylate (2-5A) synthetase are two interferon-induced and double-stranded RNA-activated enzymes which are implicated in the mechanism of action of interferon. Their distribution was undertaken here at the ultrastructural level by the immunogold procedure, following the use of specific monoclonal antibodies directed against PKR and 69- and 100-kDa forms of the 2-5A synthetase. These enzymes were detected as a pool of nonaggregated proteins scattered throughout the cell and as aggregates often associated with electron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, and the nucleus. In general, the 2-5A synthetases were present in much more lower amounts than the PKR, probably due to the difficulty of detecting traces of proteins by electron microscopy. To circumvent this, we used a human lymphoblastoid cell line overexpressing the 69-kDa form of the 2-5A synthetase. In such cells, the synthetase was then clearly observed in both the cytoplasm and the nucleus; isolated or small clusters of gold particles were numerous in the cell mainly over the RNP fibrils of the interchromatin space, nucleolus, and ribosomes. Interestingly, gold particles were also found to be associated with the membranes of nuclear envelope and rough endoplasmic reticulum probably due to the myristilated motif of this form of 2-5A synthetase. Finally, intensely labeled electron-opaque dots sometimes associated with the nuclear pore complexes were present in the nucleus and in the cytoplasm of cells which might suggest their transport from the nucleus to the cytoplasm or reciprocally through the nuclear pore complexes. These observations indicate the wider distribution of the dsRNA-activated enzymes in the cell, thus pointing out their potential implication in as yet undetermined physiological function(s) necessary for various cellular metabolic reactions.

The (2'-5')oligoadenylate synthetase represents a family of interferon-induced proteins which when activated by double-stranded (ds)RNA polymerizes ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)n, where n > 1, which for convenience are referred to as 2-5A. We studied here the influence of pH, dsRNA concentration and time on oligomeric composition of 2-5A synthesized by purified 69-kDa and 100-kDa isoforms of (2'-5')oligo(adenylate) synthetase. In optimal conditions for activity, the 69-kDa form synthesized higher oligomers of 2-5A molecules whereas the 100 kDa form synthesized preferentially dimeric molecules, which are known not to be functional for the activation of RNase L. This difference does not reflect a differential affinity of the enzymes for the preformed 2-5A dimer, which is found to be a very poor substrate for both enzymes. This latter strongly suggests that the mechanism of elongation is more likely processive. Moreover, we show that both isoforms have efficient nucleotidyl-transferase activity and provide evidence that, in optimized conditions, GTP can be used alone as substrate by these enzymes to generate pppG2'p5'G. Our results clearly demonstrate that the 69-kDa and 100-kDa forms of (2'-5')oligoadenylate synthetase manifest various differential catalytic activities, and favor the hypothesis that these enzymes might have other functions in the cell besides those in the 2-5A system.

Adenosine deaminase (ADA) and the HIV-1 transactivator protein Tat have been reported to bind to human CD26, also known as dipeptidyl peptidase IV (DPP IV). In order to demonstrate the specificity of such binding under native conditions of CD26, i.e., when expressed on the cell surface, we established murine cell lines expressing transfected human CD26, either wild-type or mutated at its serine-630, which inactivates the DPP IV activity. This experimental system is advantageous since murine ADA does not bind human CD26, whereas human and bovine ADA bind. Consequently, murine cell clones expressing either the wild-type or mutated form of human CD26 were found to bind specifically bovine 125I-labeled ADA with a high affinity (KD = 12 +/- 2 nM and 11 +/- 4 nM, respectively). No specific binding of 125I-labeled ADA was observed to murine clones not expressing human CD26. The binding of 125I-labeled ADA to CD26 was further characterized by the use of monoclonal antibodies specific to human CD26. The results obtained were in accord with those reported previously using other experimental models. These observations indicated that the murine cells expressing human CD26 provide a highly suitable model to investigate the potential binding of HIV-1 Tat to CD26. In contrast to previously published results, however, we could not demonstrate a specific interaction between Tat and human CD26. The 125I-labeled ADA-specific binding to human CD26 was not affected by Tat, even at concentrations which induced cell death. Similarly, the binding of several monoclonal antibodies to human CD26 was not modified by the addition of Tat. More significantly, Tat binding to different murine cell clones (human CD26 negative or positive) was found not to be correlated with the expression of human CD26. Finally, the toxic effect of Tat on the growth of different murine cell clones was independent of human CD26 expression. Taken together, these observations further confirm the specific binding of ADA to human CD26 and point out that CD26 is not the target of HIV-1 Tat protein.

Four isoforms of 2',5'-oligoadenylate (2-5A) synthetase have been identified (40, 44, 69, and 100 kD). The 40- and 44-kD enzymes are encoded by the same gene, probably different from the genes encoding the larger isoforms. In this study, induction of the 100- and 69-KD (p100, p69) isoforms in different individuals and in different cell types was investigated after treatment with recombinant human interferons (IFN): IFN-alpha 2, IFN-beta ser, or IFN-gamma. The p69 and p100 isoforms were quantitated in cell extracts on Western blots using specific polyclonal antibodies, or their activity was measured after purification of cell extracts on immunoaffinity columns. The p69 and p100 isoforms were differentially induced in Daudi, fibroblast, and colon adenocarcinoma cells treated with IFNs. Considerable individual variations in both basal and induced levels of p69 and p100 were observed in peripheral blood mononuclear cells from normal donors cultured with IFNs in vitro, and from cancer patients treated with IFN-alpha 2 or with IFN-beta ser. These results demonstrate that the p69 and p100 isoforms are present in vivo in peripheral blood mononuclear cells and their level is increased following IFN administration. Furthermore, both the in vivo and in vitro observations indicate that the expression of these enzymes is specific to each cell type and varies among individuals.

The existence of three distinct forms of 2-5A synthetase, p40-p46, p69, and p100, has been established in interferon-treated human cells. The expression of these enzymes varies according to the cell type studied, and their properties are relatively different. By the use of polyclonal antibodies specific to p69, we have cloned several cDNAs which identify four interferon-induced RNAs of 5.7, 4.5, 3.7, and 3.2 kilobases (kb). Analysis of the nucleotide sequence of three full-length cDNAs (5.6, 3.1, and 2.9 kb) revealed that they have a common open reading frame of 683 amino acids with different 3' termini; cDNAs 5.6 and 3.1 have an extension of 4 amino acids, whereas cDNA 2.9 has an extension of 44 amino acids. In vitro transcription-translation of cDNAs 3.1 and 2.9 kb generated proteins of 69 and 71 kDa, respectively. Both proteins bind a monoclonal antibody specific for p69 and can synthesize 2-5A. The deduced amino acid sequence of p69 revealed that it can be divided into two homologous and adjacent domains, each sharing strong homology to the first 346 amino acids common to the two isoforms of the small 2-5A synthetase (p40, p46). These results suggest that p69 might have two functional catalytic domains required for 2-5A synthetase activity and favor the hypothesis that its gene might have derived from the fusion of two ancestral genes analogous to the small 2-5A synthetase gene.

Three major forms of 2',5' oligoadenylate (2-5A) synthetase are induced in interferon (IFN)-treated human cells: 40-46, 69, and 100 kD. Here we studied the binding and activation of the 69- and 100-kD forms to single-stranded (ss) or double-stranded (ds) RNAs. The 69- and 100-kD form enzymes purified by immunoaffinity chromatography, were shown to be activated by synthetic dsRNAs poly(I).poly(C) or poly(A).poly(U) whereas ssRNAs poly(I), poly(C), poly(A), poly(U), and poly(G) had no effect. Both enzymes were also partially purified by binding to dsRNA or ssRNA-Sepharose. The synthetases bound to dsRNA Sepharose were partially activated but required the addition of soluble poly(I).poly(C) for maximal activity. The synthetases bound to ssRNA-Sepharose manifested no activity but became activated in the presence of soluble dsRNA, poly(I).poly(C). However, activation of such ssRNA-bound enzymes by dsRNA did not result in their dissociation from the ssRNA-Sepharose. These results indicate the presence of different polynucleotide binding sites on the 69- and 100-kD forms of 2-5A synthetase: a specific dsRNA binding site essential for activation and another polynucleotide binding site or sites which, although not specific, might be important for the optimal conformation of these proteins in cells.

Using specific monoclonal and polyclonal antibodies, the induction, synthesis and subcellular localization of 69- and 100-kDa forms of 2',5'-oligoadenylate (2-5A) synthetase (p69 and p100) were investigated in alpha-interferon-treated human HeLa and Daudi cells. Although both p69 and p100 were induced by interferon, there were significant differences in the interferon dose-response and the kinetics of synthesis of each protein in these cell lines. Both proteins are localized mainly in the cytoplasm. However, immunoenzymatic staining analysis of interferon-treated cells indicated that a proportion of p69 is concentrated around the nuclei and the rest is distributed in a specific pattern in the cytoplasm whereas p100 is found in a diffuse state in the cytoplasm. In accord with its association to cell membranes, p69 is myristilated. The [35S]cysteine and [3H]myristate-labeled p69 preparations were resolved by two-dimensional gel isoelectric focusing with pI values in the pH range of 7.0-8.0 and 7.0-7.5, respectively. These observations suggested the existence of two forms of p69 which might be complexed together as a dimer. In favor with a dimeric form of p69, in gel filtration experiments the peak of p69 was routinely found in fractions corresponding to a molecular mass of 160 kDa whereas p100 was recovered as a monomer. Taken together, these results indicate that p69 and p100 have distinct characteristics and that their expression is a specific property of each cell type in response to interferon.

Recently, the existence of 40-, 46-, 69- and 100- kDa forms of 2',5'-oligoadenylate (2-5A) synthetase have been established in interferon-treated human cells. Using monoclonal antibodies specific for 69- and 100- kDa forms of 2-5A synthetase, we purified these proteins by immunoaffinity chromatography and raised murine polyclonal antibodies. All immunized mice developed antibodies (anti-69 or anti-100 kDa form) which were characterized by their capacity to immunoprecipitate [35S] cysteine labeled proteins from interferon-treated cells or identify these proteins by electrophoretic transfer immunoblot analysis of extracts from control and interferon-treated cells. The 69 and 100 kDa 2-5A synthetases were induced in different types of human cells, such as Daudi, BJAB, HeLa and differentiated HL-60 cells. These enzymes were not detectable nor induced in MRC5 and undifferentiated HL-60 cells.